Many bacteria utilize small molecule or peptidic signals to assess their local population densities in a process termed quorum sensing (QS). [1-3] This chemical signaling process effectively allows bacteria to “count” themselves and behave as a group at high cell number. While the specifics may vary between species, QS circuits share general organizing principles: bacteria produce, secrete, and detect signal molecules referred to as autoinducers. At high population densities in a given environment, the autoinducers will reach a sufficiently high concentration to bind and activate their cognate (intracellular or extracellular) receptors. Signal:receptor binding then alters the expression of genes involved in bacterial group behaviors, such as swarming, sporulation, bioluminescence, conjugation, biofilm formation, and virulence factor production. [4-6] These phenotypes can have widespread and sometimes devastating effects on human health, agriculture, and the environment.[7, 8] For example, pathogenic bacteria utilize QS to launch synchronized attacks on their hosts only after they have achieved a high cell density, thereby overwhelming the defense mechanisms of the host.[9-11] As several prevalent human pathogens (e.g., Staphylococcus aureus) use QS to control virulence, QS has received considerable recent attention as a new anti-infective target.[12] There has been significant interest in the development of non-native ligands (e.g., small molecule and peptides) capable of blocking QS pathways. In contrast to antibiotics, which target bacterial pathways that are essential for survival, QS antagonists provide an alternative anti-infective therapy that does not place selective pressure on the bacterial population to develop resistance.[13, 14] This is especially important in the case of S. aureus, which rapidly develops resistance to antibiotics, including resistance to the once last-resort antibiotic vancomycin.[15]
S. aureus is a Gram-positive bacterium that uses QS to establish both acute and chronic infections.[16, 17] This pathogen produces an impressive arsenal of virulence factors, including tissue-degrading enzymes, immune evasion factors, and pore-forming toxins (hemolysins), all of which are regulated by the accessory gene regulator (agr) QS system.[18-20] The agr system has four components, termed AgrA-D, as illustrated in FIG. 1A, and is centered on the autoinducing peptide (AIP) QS signal. AgrB is an integral membrane endopeptidase that converts the precursor of the AIP signal, AgrD, to the mature AIP. This conversion involves cyclization of AgrD via a cysteine sulfhydryl group and its C-terminus to form the AIP as a 16-atom thiolactone macrocycle with an N-terminal exocyclic tail (shown in FIG. 1B). AgrB is also involved in the secretion of AIP across the cell membrane. Once a threshold concentration of AIP is reached in a given environment, the AIP ligand binds to its target receptor AgrC, a transmembrane histidine kinase. The AIP:AgrC complex acts to phosphorylate the response regulator, AgrA. Phosphorylated AgrA then binds to the P2 and P3 promoters to autoinduce the agr system and upregulate RNAIII transcription.[21] RNAIII thus represents the main effector of the agr system and regulates the expression of many virulence factors and surface proteins associated with biofilm production.[22]
There is a hypervariable region within the S. aureus agr operon that has led to the classification of four agr specificity groups of S. aureus (I-IV) with distinct AIP and AgrC sequences.[23-25] The structures of the four AIP signals (I-IV) are shown in FIG. 1B. The four AIP signals have a conserved 16-atom thiolactone macrocycle, and AIPs I and IV share a nearly identical primary sequence, while AIP-II and AIP-III have more dissimilar primary sequences.
The four different agr groups have been correlated with specific disease types: group-I and -II are associated with the majority of invasive infections,[26-28] while group-IV is considered rare and limited to exfoliative toxin-related syndromes8 26] Group-III S. aureus has recently been reported to be the most abundant group in nasal carriage cases and to be predominately responsible for toxic shock syndrome (TSS) in humans.[26, 27] Toxic shock syndrome toxin-1 (TSST-1) is the causative agent all cases of menstrual TSS and most cases of nonmenstrual TSS.[26, 29] Notably, TSST-1 production is directly regulated by the agr-III QS system (FIG. 1A).[18, 29, 30] Methods to inhibit the agr-III system in S. aureus could provide new insights into and therapeutic strategies for this deadly disease.
QS is dependent on autoinducer:receptor binding, and the development of chemical agents capable of blocking this binding event have been a focus of considerable research.[31-33] Both small molecules and macromolecules have been utilized to block native autoinducer binding, largely in Gram-negative bacteria.[34, 35] Such abiotic agents can be useful in anti-infective treatments.[36-38] The development of small molecules that affect AgrC signaling in S. aureus has proceeded more slowly.
Janda et al. [35, 44] have recently reported a complimentary strategy based on antibodies that sequester the AIP ligand away from AgrC and effectively “quench” QS in group-IV S. aureus. McCormick and co-workers have reported that naturally occurring cyclic dipeptides produced by Lactobacillus reuteri can modulate the agr system in S. aureus.[29]
Early studies of the AgrC systems reported that each of the native AIPs were capable of cross-inhibiting the other three, non-cognate receptors.[23, 45-47] This activity was suggested to provide each group some competitive advantage when establishing an infection, and to explain in part the predominance of a single S. aureus group in many infection types.[23, 48] Given the prevalence of group-I and -II systems in clinically relevant infections, AIP-I and AIP-II have so far received the most scrutiny for the design of AgrC modulators.[24, 45, 46, 49-52]
Studies by Muir, Novick, Williams, and co-workers examined the structure activity relationship (SAR) of AIP-I and AIP-II, [53-55] and reported certain non-native mimetics of these peptides that were capable of inhibiting both their cognate and non-cognate AgrC receptors in S. aureus. There are two components to AIP:AgrC interactions: initial recognition and induction of an allosteric response that drives activation. The core AIP-II macrocycle was reported to be important for recognition because linear native peptides and mimetics of AIP-II were completely inactive.[45] In addition, these studies reported that the two structural elements of AIPs, the macrocycle and the exocyclic tail, are responsible for AgrC:AIP recognition and AgrC activation, respectively. [55] Interactions of AIPs with non-cognate AgrC receptors were reported to be inhibitory, namely through AIP:AgrC recognition, alterations to the exocyclic tails did not significantly affect cross-inhibition. The AIP macrocycle alone is reported sufficient for cross-group inhibitory activity. Within the AIP-I and AIP-II macrocycles, the hydrophobic residues at the C-termini were reported to be essential for cognate and non-cognate AgrC recognition.[24, 45, 49] Consistent with these observations, Muir et al. reported several potent and global inhibitors of all four AgrC receptors.[49] The most active inhibitor was reported to be a truncated version of AIP-I that lacked an exocyclic tail and had an aspartic acid to alanine mutation in the macrocyclic core (tAIP-I D2A):
Peptidomimetics are a powerful tool for studying and understanding SARs of peptides and proteins. [67] Modifications can be introduced to gather information regarding the importance of specific amide and hydrogen bonds within a peptide backbone and to identify conformational restrictions and structural elements important to overall activity while simultaneously enhancing pharmacological properties. [68-73] Peptidomimetics, with enhanced metabolic stability and permeability, can be useful as drug leads. [67, 74] For instance, N-methyl amino acids and N-substituted glycine derivatives (peptoids) can be inserted into or formed into peptides generating fully or partially N-methylated peptides, peptoids or peptide-peptoid (peptomer) hybrids which can be used to provide valuable SAR insights.

For example, Muir and co-workers [51] performed a N-methyl scan of the truncated version of AIP-II pointing to the utility of such studies for AIP SAR.
The SARs that dictate the activity of AIP-III remain largely unknown, and mimetics thereof are yet to be reported.

Molecules which affect QS in group-III S. aureus are of particular interest because of the prevalence of infections of these group-III species in human TSS.